Abstract
Thousands of proteins have been found to be modified by O-GlcNAc, a common glycosylation modification of serine and threonine residues throughout the cytosol and nucleus. O-GlcNAc is enzymatically added and removed from proteins, making it a potential dynamic regulator of cell signaling. However, compared with other posttranslational modifications like phosphorylation, relatively few O-GlcNAc-regulated pathways have been discovered and biochemically characterized. We previously discovered one such pathway, where O-GlcNAc controls the contraction of fibroblasts initiated by the signaling lipid sphingosine-1-phosphate. Specifically, we found that O-GlcNAc modification of the phosphatase MYPT1 maintains its activity, resulting in dephosphorylation and deactivation of the myosin light chain of the actinomyosin complex. Another signaling lipid that leads to contraction of fibroblasts is lysophosphatidic acid, and this signaling pathway also converges on MYPT1 and actinomyosin. We therefore rationalized that O-GlcNAc would also control this pathway. Here, we used a combination of small molecule inhibitors, 2D and 3D cell cultures, and biochemistry to confirm our hypothesis. Specifically, we found that O-GlcNAc levels control the sensitivity of mouse and primary human dermal fibroblasts to lysophosphatidic acid–induced contraction in culture and the phosphorylation of MLC and that MYPT1 O-GlcNAc modification is responsible. These findings further solidify the importance of O-GlcNAc in regulating the biology of fibroblasts in response to procontractile stimuli.
Highlights
Opposing enzyme termed O-GlcNAcase (OGA) [4, 5]
To determine whether overall O-GlcNAc levels alter the sensitivity of fibroblasts to lysophosphatidic acid (LPA)-induced contraction, we took advantage of two small molecule inhibitors: 5SGlcNAc (OGT inhibitor) [45] and Thiamet-G (OGA inhibitor) [46]
We found that O-GlcNAc levels control the sensitivity of fibroblasts to LPA in the medium, setting up this modification as a dimmer switch that regulates the concentration of LPA required to drive contraction
Summary
Opposing enzyme termed O-GlcNAcase (OGA) [4, 5]. These properties, combined with the fact that O-GlcNAc occurs on serine and threonine residues that can be occupied by phosphorylation [11], make O-GlcNAc a potentially critical signaling regulator. We show that O-GlcNAc controls LPA-mediated contraction of primary human dermal fibroblasts in 3D collagen cultures. We first treated NIH3T3 mouse fibroblasts in 2D cell culture with 5SGlcNAc (200 μM) for 16 h, resulting in a loss of O-GlcNAc modification compared with the DMSO vehicle (Fig. S1A).
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